It is widely observed that biological systems are phenotypically robust to both genetic and environmental perturbation. However, it is also observed that a clonal population of cells will invariably display heterogeneous phenotypes. Such heterogeneity is at the root of several human health issues including antibiotic resistance in pathogens, aging, cancer progression, and the response of cancer cells to drug therapies. The work described in this proposal will seek to elucidate the mechanisms that underlie phenotypic heterogeneity. It has been proposed that noise in gene expression is an important source of phenotypic variability, but it has been difficult to test this hypothesis directly. Work n our lab has identified a set of yeast mutants that display a high degree of variability in morphological phenotypes. This set is enriched for genes with roles in chromatin regulation, such as SNF6. Interestingly, SNF6 mutants have also been shown to exhibit increased transcriptional noise. This seems to support the hypothesis that noise in gene expression is associated with phenotypic variation; however, several genes that are known cooperate with SNF6 in chromatin remodeling do not increase morphological variability when mutated. To clarify the relationship between transcriptional noise and phenotypic variation, we must measure these phenomena together, in a variety of genetic backgrounds. The primary objective of this project is to elucidate the mechanisms that cause phenotypic heterogeneity in a population of isogenic cells. We hypothesize that noise in gene expression underpins many aspects of phenotypic variability. To test this hypothesis, we will use the SWI/SNF chromatin remodeling complex as a case study. We will first measure the phenotypic variability of mutants in each of the 12 subunits of the SWI/SNF complex. We will expand upon previous investigations by measuring heterogeneity in cell morphology and growth rate. Additionally, we will measure the expression of fluorescent reporter proteins to determine the amounts, and types, of transcriptional noise in each of these mutants. Many of the proposed experiments will utilize a system of high-throughput microscopy and automated image analysis that has been developed in our lab. Using this system, we will image hundreds of thousands of cells which are fixed, stained, and deposited in glass-bottom 96-well plates. We will then quantify the morphological variability present in each mutant background. Additionally, we will utilize this system to monitor the growth of individual micro-colonies and determine the distribution of growth rates in each genotype. Finally, we will use fluorescent reporter proteins, driven by a variety of promoters, to determine the effect that each SWI/SNF mutant has on gene expression noise. The data generated in these experiments will allow us to determine whether stochastic gene expression is a requisite for phenotypic heterogeneity or whether variability in phenotype can arise by other means. PUBLIC HEALTH RELEVANCE: It is widely believed that biological phenotypes are determined solely by genetics, the environment, and gene- environment interactions. However, isogenic populations of cells will invariably display a range of phenotypes, even in highly homogenous environments. We will test the hypothesis that stochastic fluctuations in gene expression contribute to phenotypic variability.